Amplification of DNA by polymerase chain reaction (PCR) is a technique fundamental to molecular biology. Nucleic acid analysis by PCR requires sample preparation, amplification, and product analysis. Although these steps are usually performed sequentially, amplification and analysis can occur simultaneously. DNA dyes or fluorescent probes can be added to the PCR mixture before amplification and used to analyze PCR products during amplification. Sample analysis occurs concurrently with amplification in the same tube within the same instrument. This combined approach decreases sample handling, saves time, and greatly reduces the risk of product contamination for subsequent reactions, as there is no need to remove the samples from their closed containers for further analysis. The concept of combining amplification with product analysis has become known as “real time” PCR. See, for example, U.S. Pat. No. 6,174,670.
Monitoring fluorescence during each cycle of PCR initially involved the use of ethidium bromide. (Higuchi, R., Dollinger, G., Walsh, P S, and Griffith, R., Simultaneous amplification and detection of specific DNA sequences, Bio/Technology 10 (1992) 413-417; Higuchi, R., Fockler, C., Dollinger, G., and Watson, R., Kinetic PCR analysis: real time monitoring of DNA amplification reactions, Bio/Technology 11 (1993) 1026-1030). In that system fluorescence is measured once per cycle as a relative measure of product concentration. Ethidium bromide detects double stranded DNA; if template is present fluorescence intensity increases with temperature cycling. Furthermore, the cycle number where an increase in fluorescence is first detected increases inversely proportionally to the log of the initial template concentration. Other fluorescent systems have been developed that are capable of providing additional data concerning the nucleic acid concentration and sequence.
In kinetic real time PCR, the formation of PCR products is monitored in each cycle of the PCR. The amplification is usually measured in thermocyclers which have additional devices for measuring fluorescence signals during the amplification reaction.
A prerequisite for the performance of real time PCR is the availability of synthetic oligonucleotides which can act as amplification primers and as hybridization probes. Synthetic (deoxy)-oligonucleotides are usually prepared on a solid phase with the aid of phosphoramidite chemistry. Glass beads having pores of a defined size (abbreviated in the following as CPG=controlled pore glass) are usually used as the solid phase. The first monomer is bound to the support via a cleavable group such that the oligonucleotide can be cleaved after the solid phase synthesis is completed. The first monomer additionally contains a transiently protected hydroxyl group, whereas dimethoxytrityl (DMT) being usually used as the protective group. The protective group can be removed by acid treatment. Then 3′-phosphoramidite derivatives of (deoxy)-ribonucleosides that are also provided with a DMT protective group are then coupled in a cyclic process to each successive reactive group after is has been freed of the DMT protective group. The monomers contain additional permanent protecting groups, i.e. OH protecting groups and base protecting groups which are cleaved after the synthesis is completed. For review, see: Oligonucleotide synthesis: a practical approach. Ed. M. J. Gait, IRL Press Oxford, 1988.
Oligonucleotide synthesizers for high throughput oligonucleotide synthesis are commercially available. “Dr Oligo” (BiolyticLab Performance Inc), “Mermade” (Bioautomation) and “Polyplex” (Genmachines Genomic Solutions Inc) are synthesizer using microtiterplates with a frit in each bottom of a well as synthesis platform. The frit retains the solid support inside the well. Alternatively a 96 position rack is used in which in each position fits a synthesis column (commercially available from Applid Biosystems) such columns contain two frits one at the bottom and the other on the top of the solid support. Alternatively, CPG Frits could be used (available from CTGen)
Such Synthesizers are described in U.S. Pat. No. 5,368,823; U.S. Pat. No. 5,541,314; EP 0 683 790; US 2003/113,236; US 2003/086,829; US 2002/176,811 and Rayner, Simon, et al., Genome Research 8(7) (1998); Peck, Konan, Nucleic Acids Res. 30 (2002) 18 e93
However, there is no integrated system available which enables for an automated PCR reaction, which includes synthesis of the required oligonucleotides.